【作者】 王磊；

【导师】 李保强；

【作者基本信息】 哈尔滨工业大学 ， 材料学， 2017， 博士

【摘要】 在组织工程与可再生医学当中,水凝胶支架的构建是体外组织培养的重要步骤之一。在水凝胶的研究中,分子的设计合成、功能化及其降解行为的监控尤为重要。形成水凝胶的天然高分子材料中,壳聚糖由于具有生物可降解性、生物相容性以及氨基赋予的生物活性等特点。由于壳聚糖无法UV交联以及不具有温度响应性的缺点,使其在组织工程,药物释放领域的应用受到限制。本研究以设计合成UV交联壳聚糖以及进一步赋予其温度响应性为目标,针对性设计合成两种壳聚糖衍生物,并实现快速UV固化以及温度响应性。使用碳纳米点作为荧光探针实现UV交联壳聚糖水凝胶的可视化降解监测。针对壳聚糖存在的中性pH无法水溶以及无法UV交联的缺点,采用一步N-酰化合成UV交联壳聚糖―甲基丙烯酰基壳聚糖(N-MAC)。随着甲基丙烯酸酐与壳聚糖氨基比例的增加,甲基丙烯酰基取代度增加。取代度增加到28.4%,中性pH溶液中的溶解浓度高达27.6 mg/mL。UV交联壳聚糖可通过快速UV辐照(30 s)实现固快速化。UV交联壳聚糖水溶液及其水凝胶都具有较好的细胞相容性。通过UV光刻方法制备出多种图案化微凝胶(包括规则图案以及复杂徽标)。此外UV交联壳聚糖还可通过皮下注射方式实现快速皮下UV固化,其水凝胶在体内降解不会引起慢性炎症反应,具有较好体内生物相容性。将羟基磷灰石负载于UV壳聚糖中制备出复合水凝胶,可修复临界尺寸的颅骨缺损。缺损修复效果与羟基磷灰石组分呈正相关性。针对UV交联壳聚糖缺少温度响应性,依次采用自由基接枝与N-酰化两步反应合成温敏UV交联壳聚糖—异丙基丙烯酰胺接枝甲基丙烯酰基壳聚糖。温敏UV交联壳聚糖水凝胶具有温度引发的可逆的体积收缩与膨胀性能。可通过温敏单元NIPAM加入量调控转变温度(LCST)以及水凝胶溶胀性能。将具有光热效应的碳纳米片负载于温敏水凝胶中,可实现近红外光(NIR)引发的水凝胶体积收缩。将模型药物多柔比星(DOX)负载其中,可实现NIR引发DOX的按需释放行为。针对碳纳米点荧光探针存在合成产率低(小于50%)的缺点,分别将含有碳碳双键的壳聚糖与含有碳碳双键小分子/壳聚糖前驱体通过水热碳化法合成氮掺杂碳纳米点。其中碳碳双键的含量与合成产率呈现正相关性,最高产率可达85.9%。碳纳米点由于氮元素的掺杂使得其具有较宽的发射波长(400~550nm)。当碳纳米点作为金属离子传感器检测Hg2+,其检出限低至80 n M。基于荧光图像法结合MATLAB软件以及手机APP可实现快速、便携以及低成本Hg2+检测。碳纳米点荧光探针容易在斑马鱼的肝脏、卵黄以及消化系统富集,并可通过消化系统代谢出去,具有较好的体内生物相容性。此外研究发现碳纳米点通过下调内源与外源ROS,在体内起到氧化应激保护作用。针对有机荧光探针存在高光致漂白以及生物相容性差的缺点,采用碳纳米点作为荧光探针,赋予水凝胶以红色荧光发射。作为新型荧光探针,碳纳米点具有低光致漂白以及良好的生物相容性的优点,并可实现同一样品的长期稳定的荧光追踪。应用荧光图像衰减的方法可实现体外/体内水凝胶降解的定量监测。更多还原

【Abstract】 Hydrogel scaffoldsplay an important role in tissue engineering and regenerative medicine.Serving as one of biomaterial scaffolds,hydrogels are generally regarded as biocompatible materials because their high water content and soft nature render them similar to natural extracellular matrices and minimize tissue irritation and cell adherence.Furthermore,their porous structure,along with their water content,is suitable properties to accommodate high loads of water-soluble compounds,such as therapeutically active proteins and peptides.Chitosan is a biodegradable and natural biomaterial with amino groups,which has been widely used in tissue engineering and drug delivery owing to its biodegradability by lysozyme,biocompatibility,antibacterial activity and hemostatic ability.However,there are some challenges associated with chitosan for its applications in tissue engineering,such as poor solubility in neutral pH solution and lack of UV crosslinking and thermo-responsive ability.So we facilely designed a hydrosoluble,UV crosslinkable chitosan derivative by N-acylation and further endowwith thermo-responsive ability.In this doctoral dissertation,we employed carbon nanodots(CNDs)with low photobleaching,red emission and good biocompatibility as fluorescent indicator for real-time and non-invasive visual in vitro/in vivo degradation of hydrogels.The limit of existing chitosan includes: 1.could not dissolve in neutral pH solution;2.could not be UV crosslinked.In this study,we facilely synthesized a hydrosoluble,UV crosslinkable and injectable chitosan by single-step N-acylation.The degree of substitution(DS)of methacryloyl groupswas positively correlated to the ratio of MA used in acylation reaction,which could be controlled by regulating ratio of anhydride to amino groups.The solubility of N-MAC in neutral pH solution increased to 27.6 mg/m L with DS increasing to 28.4%.N-MAC hydrogel was obtained via photopolymerization of carbon–carbon double bonds under(30s)UV irradiation.UV-crosslinkable chitosan exhibits good biocompatibility.Patterned cell-laden microgels with on-demand regular geometric shapes and complex logos were fabricated via lithography.Injectable and rapid transdermal curing hydrogels were developed via skin-penetrable UV-crosslinking strategy within mice subcutaneous space.UV-crosslinkable chitosan hydrogels stimulated a relatively slight acute inflammatory response but did not transformed into chronic inflammation,and could afford good biocompatibility.Hydroxyapatite(HA)/UV-crosslinkable chitosan hydrogelwas prepared by mixing chitosan with HAand sequentiallyapplying for UV irradiation.The high HA content in composite hydrogel benefited the acceleration of bone regeneration.To endow UV-crosslinkable chitosan with thermo-responsive ability,a novel UV-crosslinkable and thermo-responsive chitosan was designed by grafting with poly N-isopropylacrylamide(PNIPAM)and acetylation of methacryloyl groups.The thermo-responsive unit PNIPAM endowed chitosan hydrogel with temperature triggered volume shrinkage and reversible swelling/de-swelling behavior.DOX release rate was accelerated and approximately 40 times higher than that from non-irradiated hydrogels.The UV-crosslinkable and thermal-responsive hydrogel served as in situ forming hydrogel-based drug depot is developed for NIR-triggered localized on-demand release.To date,synthetic yield of CNDs via hydrothermal carbonization is quite low(<50%).We report here the synthesis of CNDs derived from chitosancontaining carbon-carbon double bond and small molecule containing carbon-carbon double bond/chitosan.The CNDs exhibited a constant increase in synthetic yield(increase to 85.9%)and content dependent feature.Hence the concept of carbon-carbon double bonds boosting ultrahigh-yield synthesis of CNDs provides a promising strategy to be employed as carbonaceous nanodrug aiming at preventing and curing ageing and age-related diseases.These CNDs exhibited a high quantum yield and wide emission wavelengths(400~550 nm)as a consequence of nitrogen incorporation.We further demonstrate applications of CNDs as probes for heavy metal ion detection.The CNDs offered potential as mercury ion sensors with detection limit of 80 n M.A smartphone APP based on CNDs was developed providing a portable and low cost detection platform for detection of heavy metal ions contamination.CNDs could be uptaked and metabolism mainlythrough the digestive system in zebrafish.The CNDs exhibit outstanding protective effect against oxidative stress viadown-regulating exogenous and endogenous ROS generation.We employed CNDs with low photobleaching,red emission and good biocompatibility as fluorescent indicator for visual in vitro/in vivo degradation of hydrogels.The embedded CNDs in hydrogels did not diffuse outside in the absence of hydrogel degradation.We had acquired similar degradation kinetics between gravimetric and visual determination,and established mathematical equation to quantitatively depict in vitro degradation profile of hydrogels.Based on the in vitro data,we developed a visual platform that could quantitatively depict in vivo degradation behavior of new injectable biomaterials by real-time and non-invasive fluorescence tracking.This fluorescence-related visual imaging methodology holds great potentials for rational design and convenient in vivo screening of biocompatible and biodegradable injectable hydrogels in tissue engineering.更多还原